Abstract Directed cell migration is important in many normal and pathological processes including embryogenesis, wound healing, and tumor progression. The tensed actomyosin stress fiber (SF) network is largely responsible for generating contractile forces to establish the cell structures and shape needed for polarized migration. Over the past ten years, the field has classified SFs into three subpopulations, each differing in their connections to focal adhesions, molecular composition, and localization in a migrating cell. However, it is unclear how the mechanical properties of individual SFs in each of the subpopulations contribute to maintaining cell shape, tension, and migration. Furthermore, much of the existing knowledge on SFs has been indirectly inferred from observational studies of cells cultured on idealized two-dimensional substrates, which are not representative of the in vivo tissue microenvironment. In this fellowship proposal, I will study the mechanical properties of single SFs in each subpopulation and their contribution to generating tension to maintain cell shape and migration. I will address this in two aims, using several powerful biophysical tools. In Aim 1, I will investigate the mechanical properties of individual SFs by using femtosecond laser nanosurgery to sever single SFs to conduct loss-of-function studies. I will use fluorescence microscopy to examine changes in the redistribution of the tension released by the severed SF to the surrounding cytoskeletal network. Furthermore, I will also examine the traction forces exerted onto the extracellular matrix by each of the stress fiber subpopulations using model-based traction force microscopy. In Aim 2, I will investigate the role of the stress fiber subpopulations in cells cultured in polyacrylamide microchannels, which have been shown previously to capture important features of confined invasive migration in vivo. I am interested in studying how these complex environments affect stress fiber subpopulation formation. I will also repeat the laser nanosurgery and traction force experiments outlined in Aim 1 in cells cultured in these microchannels. Finally, I will compare the roles of the stress fiber subpopulations in both the idealized 2D substrates in Aim 1 and the microchannels in Aim 2. Through these studies, I hope to enhance the field?s understanding of how the actin cytoskeleton regulates tension and cell shape for directed migration.